P1528: GENETIC AND PHENOTYPIC CHARACTERIZATION OF HEREDITARY SPHEROCYTOSIS AND RELATED DISORDERS – DESCRIPTION OF 9 NEW GENETIC VARIANTS
Authors: F. Haeuser, H. Rossmann, A. Adenaeuer, B. Lämmle, C. Paret, K. J. Lackner, J. Faber, O. Beck
Journal: HemaSphere (2022)

Background: Erythrocyte membrane defects are a group of hereditary diseases with highly variable severity of symptoms and caused by a set of different variants of genes encoding for red cell membrane and skeleton proteins. Although the most common hereditary hemolytic disease in Caucasians, hereditary spherocytosis (HS) is underdiagnosed, because mild clinical course is common. While mostly variants of structural proteins of the erythrocyte membrane (i.e. ANK1 (ankyrin), SLC4A1 (band 3), SPTA1 (α-spectrin) or SPTB (β-spectrin)) can be causative for the phenotype, only the minority of patients have been genetically characterized so far. Aims: We aimed to develop a simple strategy to confirm the suspected diagnosis of HS using an NGS (next generation sequencing)-based approach. Methods: 12 patients with clinical and laboratory signs of HS and one patient with massive, unclear hemolysis and initially inconclusive laboratory results, were genetically and phenotypically characterized. In 12 patients, hemolytic spherocytic anemia was confirmed by blood smear, complete blood count, haptoglobin, bilirubin, osmotic resistance test, and flow cytometric eosin-5-maleimide binding test. Acquired hemolysis was excluded. In two patients, a membrane protein defect was suggested by SDS-PAGE. Patient 13 showed massive hemolytic anemia with reduced osmotic resistance, but without elevation of MCHC. No spherocytes but poikilocytes were observed. All patients were subjected to exome sequencing (NGS). Family members of the index patients were studied by Sanger sequencing. Results: In all 13 patients, pathogenic variants fulfilling ACMG-criteria were found limited to only 4 genes: ANK1, SLC4A1, SPTA1, and SPTB. In the 12 HS patients, variants were heterozygous, consistent with the predominantly dominant inheritance. The predescribed SLC4A1 variant Band 3 Bicêtre I was detected twice, a nonsense variant in ANK1 and one in SPTB once. The new variants were detected once each. We detected two splice-site variants in ANK1 as well as one in SPTB, one missense variant in SLC4A1, two nonsense variants in ANK1 as well as one in SPTB, and one frameshift variant with a premature termination codon in SPTB. One of the splice defects located in ANK1 and the nonsense variant in SPTB were de novo variants. Only in patient 13, in SPTA1 the low expression allele α-LELY was detected, located in trans to a missense variant. Such a compound heterozygosity in SPTA1 is typical for pyropoikilocytosis and is consistent with the clinical presentation of the patient. Medical history and standard laboratory tests (blood count including MCHC, blood smear, hemolysis parameters and osmotic resistance) are usually sufficient to establish the diagnosis of HS. More sophisticated methods such as SDS- and native PAGE or ectacytometry can elucidate unclear cases. However, in our cases, all relevant variants were located in just four genes, suggesting that a reduced panel of four to five genes (incl. EPB42 (band 4.2)) may be sufficient to characterize most of the patients. Summary/Conclusion: The diagnosis of HS and hereditary pyropoikilocytosis was confirmed in 12 and one patient respectively by NGS. Panel sequencing of the five most common genes is nowadays a comparatively rapid and inexpensive method to confirm the diagnosis of HS and related disorders. Time-consuming or low-specificity tests can be avoided, and family members can be easily genotyped.